1. Field of the Invention
The invention is related to the detection of NT-proBNP in biological samples from canines.
2. Description of Related Art
Heart diseases affect all non-human animals. Heart diseases generally involve the cardiac valves and the cardiac muscle. Because the heart is capable of compensating for functional impairment by working harder, heart diseases in most cases remain hidden, with the consequence that the health of the heart will deteriorate due to the increased workload on the heart. The symptoms resulting from heart diseases, such as fatigue, circulatory insufficiency, and languor, can usually be recognized when the animal's heart is no longer able to compensate for the a particular functional impairment. At this stage, the heart disease has progressed to the point that curing the disease is no longer possible.
While chronic cardiac valve and cardiac muscle changes are usually incurable, the use of medicaments can slow disease progression. Therefore, an early diagnosis for heart diseases is beneficial. Routinely, physical methods are used for this purpose, such as auscultation of the heart sounds, the recording of an electrocardiogram, and X-ray and ultrasonic examinations. These examination methods have the disadvantage that they are carried out only when already visible or audible defects of the heart are recognized. Furthermore, physical examination methods require suitable and generally expensive devices in order to carry out a respective diagnosis.
In many heart diseases, such as, e.g., heart decompensation and dilated cardiomyopathy, a peptide hormone—the so-called brain natriuretic peptide (BNP)—is secreted by heart muscle tissue. Since this hormone is produced in the heart and is increasingly produced in case of overstress and congestion, determining the BNP level in blood is a suitable means for evaluating cardiac insufficiency.
BNP as well as other natriuretic peptides play an important part in regulating water balance and blood pressure. If the cardiac wall is dilated, it secrets BNP in increasing amounts, which in turn causes an excretion of sodium and liquid via the kidneys and dilation of the blood vessels. These factors can lower the blood pressure and the filling level of the heart. BNP is synthesized by the cells of the cardiac muscle as proBNP, which is cleaved into N-terminal proBNP (NT-proBNP) and BNP. Both parts of the polypeptide are delivered to the blood and can be detected therein.
The utility of both BNP and NT-proBNP as makers for cardiac disease in veterinary patients (e.g., dogs and cats) has been demonstrated in numerous studies. For instance, BNP and NT-proBNP assays have been shown to be effective as a diagnostic test for dogs, as illustrated by two studies, which report remarkably similar sensitivity and specificity (85% and 82% respectively) for differentiating the cause of clinical signs that may be attributable to cardiac disease in dogs. See Oyama M A, et al., “Assessment of serum N-terminal pro-B-type natriuretic peptide concentration for differentiation of congestive heart failure from primary respiratory tract disease as the cause of respiratory signs in dogs”, Journal of American Veterinary Medical Association (December 2009); Boswood et al., “The diagnostic accuracy of different natriuretic peptides in the investigation of canine cardiac disease”, JSAP 2007 1-7. In cats, the clinical challenge is different, as the most common cardiac disease in cats is hypertrophic cardiomyopathy. This disease remains occult or ‘silent’ with very few clinical signs that are appreciable to the pet owner until the disease is very advanced.
A number of immunoassays for the detection of NT-proBNP are known. These assays use polyclonal or monoclonal antibodies specific for epitopes within various regions of the peptide. These methods, however, are subject to variability because NT-proBNP is further processed ex vivo by various proteases in the blood serum and plasma. Therefore, immunoassays for NT-proBNP are inherently vulnerable to inconsistency due to the ex vivo degradation of the protein over time. Therefore, samples to be tested for NT-proBNP are typically refrigerated and efforts are made to conduct sample analysis as quickly as possible following taking of the sample.
Accordingly, the inventors have identified a need in the art for a method of determining NT-proBNP that can measure NT-proBNP without regard to when a sample was taken and without cumbersome handling requirements for the sample.